LOCUS       sequence-442982-        8792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rfx Cas13d guide cloning lentiviral vector with constitutively 
            expressed puromycin resistance 2A-tagged with tagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8792)
  AUTHORS   Abbott TR, Dhamdhere G, Liu Y, Lin X, Goudy L, Zeng L, Chemparathy 
            A, Chmura S, Heaton NS, Debs R, Pande T, Endy D, La Russa MF, Lewis 
            DB, Qi LS
  TITLE     Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 
            and Influenza.
  JOURNAL   Cell. 2020 May 14;181(4):865-876.e12. doi: 
            10.1016/j.cell.2020.04.020. Epub 2020 Apr 29.
  PUBMED    32353252
REFERENCE   2  (bases 1 to 8792)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2020.04.020"; journalName: "Cell"; date: 
            "2020-05-14- 14"; volume: "181"; issue: "4"; pages: "865-876.e12"
COMMENT     Sequence Label: pSLQ5465_pHR_hU6-crScaffold_EF1a-PuroR-T2A-BFP
FEATURES             Location/Qualifiers
     source          1..8792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2620..2868
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2620..2640
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2791..2810
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2870..2905
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        2924..2959
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        3014..4192
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3245..4183
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4140..4160
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4211..4230)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4707..4727
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4817..4870
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    complement(5592..5625)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5681..6269
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5734..5754)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6152..6163)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6272..6288)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6273..6289)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6798..6978
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7040..7628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7120..7139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7799..8590,8591..8659))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8422..8441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8660..8764)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagag agggcctatt tcccatgatt
     2641 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     2701 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     2761 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     2821 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc aagtaaaccc
     2881 ctaccaactg gtcggggttt gaaacgggtc ttcgagaaga cctcaagtaa acccctacca
     2941 actggtcggg gtttgaaact ttttttttca cctatgtatc gtatcaagta ctagaactcc
     3001 ccagtgggaa ttcggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     3061 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     3121 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     3181 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     3241 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
     3301 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
     3361 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
     3421 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     3481 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     3541 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
     3601 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     3661 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
     3721 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     3781 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     3841 ctcaaaatga aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     3901 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     3961 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     4021 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     4081 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     4141 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gacgtacggc
     4201 caccatgagc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     4261 cagggctgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     4321 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     4381 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     4441 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     4501 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     4561 gcccaaggag cccgcgtggt tcctggccac agtgggtgtg tcccctgatc accagggcaa
     4621 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     4681 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     4741 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     4801 cggtgccgga tccggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa
     4861 tcctggccca atgtccgaac tgattaaaga gaacatgcac atgaaacttt atatggaagg
     4921 tacggtcgac aaccaccatt tcaaatgtac ctccgaagga gagggcaaac cctacgaggg
     4981 cacccaaacg atgagaatta aagtagtaga aggtgggccg ctgccctttg cctttgacat
     5041 tctggcaact tcttttctct acgggagtaa aacttttatc aaccatacgc aaggcattcc
     5101 agacttcttt aagcagtcat ttcccgaggg gtttacttgg gagcgggtca caacatacga
     5161 ggacggcggc gtgctgaccg ctactcagga caccagcctg caagacgggt gtttgatcta
     5221 caatgtgaag attcgaggcg taaactttac ttcaaacggc ccagttatgc agaaaaaaac
     5281 cctcggctgg gaggctttta cagagactct ttacccagcc gacggtggtt tggaaggccg
     5341 aaacgacatg gcattgaaat tggttggcgg gagccacctt atagcgaata taaagacgac
     5401 ctatcggtct aaaaagcccg ccaaaaattt gaagatgcct ggtgtatatt acgtcgacta
     5461 taggcttgag agaataaaag aagctaacaa cgaaacctat gtggagcaac atgaggttgc
     5521 agttgcacgc tactgcgatc tgccgagcaa attggggcac aagctgaatt gatgtacagt
     5581 cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt
     5641 ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     5701 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     5761 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     5821 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     5881 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     5941 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     6001 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     6061 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     6121 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     6181 ccgcggcctg ctgccggctc tgcggcctct tccgcttctt cgccttcgcc ctcagacgag
     6241 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag
     6301 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     6361 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     6421 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     6481 aagggctaat tcactcccaa ccaagacaag atatccttga tctgtggatc taccacacac
     6541 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     6601 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     6661 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     6721 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     6781 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     6841 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     6901 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     6961 tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     7021 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     7081 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     7141 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     7201 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     7261 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     7321 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     7381 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7441 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     7501 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7561 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7621 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7681 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7741 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7801 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7861 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7921 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7981 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     8041 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     8101 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     8161 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     8221 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     8281 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     8341 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     8401 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8461 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8521 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     8581 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     8641 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8701 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8761 cgcgcacatt tccccgaaaa gtgccacctg ac
//
